本文選題：牛 + 角膜基質(zhì)細胞��； 參考：《廣西醫(yī)科大學(xué)》2011年碩士論文

【摘要】：背景：高效、低成本分離出生物學(xué)功能活性高的角膜基質(zhì)細胞是開展角膜基礎(chǔ)研究的需要。目前的分離方法成本高、分離效率低,而通過培養(yǎng)達到擴增細胞數(shù)會導(dǎo)致細胞表型快速改變。本研究旨在應(yīng)用成本較低的I型膠原酶,通過改良的兩步消化法達到高效、快速、低成本分離原代牛角膜基質(zhì)細胞的目的。 目的：評價設(shè)計的I型膠原酶兩步消化法分離原代牛角膜基質(zhì)細胞的效果,并觀察體外培養(yǎng)原代牛角膜基質(zhì)細胞的形態(tài)學(xué)變化。 方法：分別用基礎(chǔ)培養(yǎng)液配制的0.5 g/L及1.0 g/L I型膠原酶以兩步酶消化法順序消化牛角膜組織,分離角膜基質(zhì)細胞,以細胞計數(shù)板進行計數(shù),檢測基質(zhì)細胞收獲效率；錐蟲藍染色法檢測收獲細胞的存活率；分離的細胞進行原代培養(yǎng),倒置顯微鏡下觀察細胞形態(tài)和生長變化；應(yīng)用Alexa488標(biāo)記的鬼筆環(huán)肽檢測原代培養(yǎng)的牛角膜基質(zhì)細胞中微絲肌動蛋白F-actin的分布。 結(jié)果：牛角膜經(jīng)兩步酶消化基質(zhì)逐步解離和降解,絕大多數(shù)細胞得以釋放和分離,分離的牛角膜基質(zhì)細胞呈圓形,透亮且大小均勻。每個角膜收獲(2.109±0.142)×106個基質(zhì)細胞,細胞存活率(91.69士3.55)%,黏附率(81.20±1.21)%。原代培養(yǎng)的牛角膜基質(zhì)細胞附壁呈樹突樣,鋪伸至星狀,融合時樹突連接呈網(wǎng)狀,其F-actin局限性分布于細胞皮質(zhì)。 結(jié)論：兩步酶消化法可使牛角膜基質(zhì)完全消化降解,具有高細胞收獲率高細胞存活率和操作簡便等特點。原代培養(yǎng)的牛角膜基質(zhì)細胞呈樹突狀,F-actin分布于細胞皮質(zhì)。
[Abstract]:Background: high-efficiency and low-cost isolation of corneal stromal cells with high biological activity is necessary for basic corneal research. The current separation methods have high cost and low separation efficiency, and the cell phenotype will change rapidly when the number of expanded cells is reached through culture. The purpose of this study was to separate primary bovine corneal stromal cells from bovine corneal stromal cells at low cost by using a low cost type I collagenase and a modified two-step digestion method. Aim: to evaluate the effect of the designed two step digestion method for the isolation of primary bovine corneal stromal cells and to observe the morphological changes of primary bovine corneal stromal cells cultured in vitro. Methods: bovine corneal tissue was digested with 0.5 g / L and 1.0 g / L I collagenase prepared from basic culture medium respectively by two-step enzymatic digestion. Corneal stromal cells were isolated and counted by cell counter to detect the harvest efficiency of stromal cells. The survival rate of harvested cells was detected by trypanosome blue staining, the isolated cells were cultured in primary culture, and the morphology and growth of the cells were observed under inverted microscope. The distribution of microfilamentactin (F-actin) in primary cultured bovine corneal stromal cells was detected by Alexa488 labeled ghost pen cyclic peptide. Results: the bovine corneal stromal cells were gradually dissociated and degraded by two steps of enzyme digestion, and most of the cells were released and separated. The isolated bovine corneal stromal cells were circular, transparent and uniform in size. The cell survival rate was 91.69 鹵3.55%, and the adhesion rate was 81.20 鹵1.21% per corneal harvest of 2.109 鹵0.142) 脳 106 stromal cells. The primary cultured bovine corneal stromal cells were dendritic, spreading to stellate, reticular dendritic junctions during fusion, and their F-actin was localized in the cortex. Conclusion: the two-step enzyme digestion method can completely degrade bovine corneal stroma, and has the characteristics of high cell harvesting rate, high cell survival rate and simple operation. Primary cultured bovine corneal stromal cells were distributed as dendritic F-actin in the cortex.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329;S823

【參考文獻】

相關(guān)期刊論文 前10條

1 于純智,趙仙桃;家兔角膜創(chuàng)傷及角膜疤痕模型復(fù)制方法[J];第四軍醫(yī)大學(xué)學(xué)報;1982年04期

2 霍霄鯤,原林,黃濤,余磊,戴景興,秦建強,夏虎;兔角膜基質(zhì)細胞的體外培養(yǎng)及生物學(xué)特性[J];第一軍醫(yī)大學(xué)學(xué)報;2002年07期

3 孫興才;細胞外基質(zhì)對角膜創(chuàng)傷修復(fù)的作用[J];國外醫(yī)學(xué).眼科學(xué)分冊;1997年02期

4 閆曉麗;;翼狀胬肉切除羊膜移植術(shù)后壞死性鞏膜炎3例[J];臨床眼科雜志;2005年06期

5 王愛麗,胡曉潔,李卿,崔磊,劉偉,曹誼林;角膜基質(zhì)細胞體外長期培養(yǎng)過程中生物學(xué)特性的改變[J];上海第二醫(yī)科大學(xué)學(xué)報;2004年04期

6 張永強,徐寧;家兔常見角膜疾病動物模型的建立[J];山西醫(yī)科大學(xué)學(xué)報;2002年04期

7 陳華，李春海，劉根明，喬伯英;CO＿2激光復(fù)制兔角膜創(chuàng)傷及瘢痕模型[J];實驗動物科學(xué)與管理;1995年04期

8 陳靜;;翼狀胬肉術(shù)后淺層鞏膜壞死12例臨床分析[J];新疆醫(yī)科大學(xué)學(xué)報;2008年08期

9 傅瑤,范先群,曹誼林;兔角膜內(nèi)皮、上皮及基質(zhì)細胞體外培養(yǎng)擴增的研究[J];眼科研究;2003年01期

10 金玲,蔡鄭東,樓月芳;內(nèi)毒素誘導(dǎo)兔角膜新生血管模型實驗研究[J];眼科研究;1998年02期

，

本文編號：1814570